Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao.

Two dimensional electrophoresis and nano-LC-MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L(-1)) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses.

Quantification of cellulose nanowhiskers sulfate esterification levels.

Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, combustion gas analysis and N(2) adsorption were used to quantify the degree of desulfation of cellulose nanowhiskers (CNWs). CNWs were produced by hydrolyzing cotton cellulose with sulfuric acid or hydrochloric acid. Hydrochloric acid treatment did not result in any cellulose chemical modification. Hydrolysis using H(2)SO(4) introduced sulfate groups onto the cellulose surface. Our results indicate that commercial cotton cellulose as received contained sulfur. The sulfur content of H(2)SO(4)-prepared CNWs was higher than that exhibited by the original cellulose due to the esterification process. Two desulfation methods, acid-catalyzed and solvolytic desulfation, have been explored to remove the sulfate groups. Neither desulfation method examined removed the sulfate groups from H(2)SO(4)-prepared CNWs completely. An estimation of surface sulfate esterification levels was made based on a model of the cellulose structure and available surface area of CNWs. According to these models, more than one third of hydroxyl groups on the surface were substituted by sulfate.

Development and validation of an assay for iodide in serum using ion chromatography with pulsed amperometric detection.

We developed and validated an ion chromatography method to assay iodide in serum sampled from rats and rabbits that had been exposed to iodomethane. Iodomethane is of interest because it is a volatile liquid pre-plant soil crop protection fumigant that has been proposed as a non-ozone-depleting alternative to methyl bromide. Serum was prepared from whole blood collected on wet ice at the time of sacrifice and kept frozen at less than -65 degrees C. For analysis, serum samples were thawed unassisted at ambient temperature. Proteins were separated from the serum samples by ultrafiltration. A 100-microl filtered serum sample was then injected into the ion chromatograph without additional sample preparation. Iodide was separated in <20 min by anion-exchange chromatography using a 25-mM nitric acid eluent. The analyte of interest was detected by pulsed amperometry using a silver working electrode. The method showed linear response over the concentration range of 100 to 5000 ng/ml iodide (r2>.998) with a lower limit of quantitation of 100 ng/ml iodide. The accuracy of the procedure, determined by spiked recovery measurements at 100 ng/ml iodide, was between 90 and 110%. A method detection limit of 20 ng/ml for iodide in serum samples was demonstrated using the method of standard additions.

Determination of residual trifluoroacetate in protein purification buffers and peptide preparations by ion chromatography.

Trifluoroacetate (TFA) is commonly used in a variety of pharmaceutical applications. Because of its toxic nature, it is important to reliably measure the effective removal of TFA. We developed an ion chromatography (IC) method to determine the concentration of residual TFA in samples found in the pharmaceutical industry. A high-capacity anion-exchange column was used to separate trace trifluoroacetate from an excess of chloride, phosphate, and other anions without the need for sample preparation. TFA was detected by suppressed conductivity. A method with four KOH eluent step changes was optimized and reproducibly executed using automated generation of the KOH eluent. We used this method to determine TFA in the following samples: a phosphate-buffered saline (PBS), an acetate-buffered saline containing protein, and a commercial peptide. The method detection limits for TFA in these samples were all less than 90 ng/ml.

Monitoring trace anion contamination in disk drive components.

Ion chromatography was used to determine trace anionic contamination on the surface of hard disk drive components. These contaminants can have a detrimental effect on device reliability and yield. Disk drive components were soaked in deionized water and these extracts were analyzed for anions. The anions fluoride, acetate, formate, acrylate, methacrylate, chloride, nitrite, bromide, nitrate, benzoate, sulfate, oxalate, phthalate and phosphate were separated on a high-performance anion-exchange column and determined at concentrations less than 1 microg/l with suppressed conductivity detection. The extract solutions were analyzed either by injecting 1 ml or by preconcentrating 5 ml. We evaluated the performance of both methods.